Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.     

Isolation of two high-molecular-mass proteinases from human erythrocytes

Two forms of a neutral--alkaline high-molecular-mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE-cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE-cellulose chromatography. Both enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23-32 kDa). Both enzymes show a proteinase activity towards casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl-valyl-leucyl-arginyl-4-methoxy-2-naphthylamide and benzyloxycarbonyl-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol-group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity.     

Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9.

Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subsite preferences of pepstatin-insensitive carboxyl proteinases from prokaryotes: kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase

Kumamolysin, a carboxyl proteinase from Bacillus novosp. MN-32, is characterized by its thermostability and insensitivity to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3-(p-nitro-phenoxy)propane. Here, its substrate specificity was elucidated using two series of synthetic chromogenic substrates: P(5)-P(4)-P(3)-P(2)-Phe*Nph (p-nitrophenylalanine: *cleavage site)-P(2)'-P(3)', in which the amino acid residues at the P(5)-P(2), P(2)' and P(3)' positions were systematically substituted. Among 74 substrates, kumamolysin was shown to hydrolyze Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu most effectively. The kinetic parameters of this peptide were K(m) = 41+/-5 microM, k(cat) = 176+/- 10 s(-1), and k(cat)/K(m) = 4.3+/-0.6 mM(-1) x s(-1). These systematic analyses revealed the following features: (i) Kumamolysin had a unique preference for the P(2) position. Kumamolysin preferentially hydrolyzed peptides having an Ala or Pro residue at the P(2) position; this was also observed for the pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4 [J-4; Shibata et al. (1998) J. Biochem. 124, 642-647]. Other carboxyl proteinases, including Pseudomonas sp. 101 pepstatin-insensitive carboxyl proteinase (PCP) and Xanthomonas sp. T-22 pepstatin-insensitive carboxyl proteinase (XCP), preferred peptides having hydrophobic and bulky amino acid residue such as Leu at the P(2) position. (ii) Kumamolysin preferred such charged amino acid residues as Glu or Arg at the P(2)' position, suggesting that the S(2)' subsite of kumamolysin is occupied by hydrophilic residues, similar to that of PCP, XCP, and J-4. In general, the S(2)' subsite of pepstatin-sensitive carboxyl proteinases (aspartic proteinases) is hydrophobic in nature. Thus, the hydrophilic nature of the S(2)' subsite was confirmed to be a distinguishing feature of pepstatin-insensitive carboxyl proteinases from prokaryotes.     

Effect of substrate residues on the P2' preference of retroviral proteinases.

The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.     

The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.     

New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.     

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.     

Role of tryptophan hydroxylase phe313 in determining substrate specificity

The active site residue phenylalanine 313 is conserved in the sequences of all known tryptophan hydroxylases. The tryptophan hydroxylase F313W mutant protein no longer shows a preference for tryptophan over phenylalanine as a substrate, consistent with a role of this residue in substrate specificity. A tryptophan residue occupies the homologous position in tyrosine hydroxylase. The tyrosine hydroxylase W372F mutant enzyme does not show an increased preference for tryptophan over tyrosine or phenylalanine, so that this residue cannot be considered the dominant factor in substrate specificity in this family of enzymes.     

Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display

We constructed a random library of hexapeptides displayed on the surface of bacteriophage T7 to determine the substrate specificity of proteinases. The phage-displayed library was subjected to repeated rounds of biopanning with native implantation serine proteinase and recombinant human kallikrein-related peptidase 6 (KLK6) followed by selection and identification of putative substrates. For both enzymes, the results obtained demonstrate a preference for arginine and lysine at multiple positions in the recognition cleavage motif, confirming their previously reported trypsin-like substrate specificity. In the case of KLK6, there is also a pronounced presence of tryptophan within the cleaved peptide sequences, indicating its potential dual substrate specificity, acting as both a trypsin and chymotrypsin-like enzyme.     

Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Three-dimensional model of a substrate-bound SARS chymotrypsin-like cysteine proteinase predicted by multiple molecular dynamics simulations: catalytic efficiency regulated by substrate binding

Severe acute respiratory syndrome (SARS) is a contagious and deadly disease caused by a new coronavirus. The protein sequence of the chymotrypsin-like cysteine proteinase (CCP) responsible for SARS viral replication has been identified as a target for developing anti-SARS drugs. Here, I report the ATVRLQ(p1)A(p1')-bound CCP 3D model predicted by 420 different molecular dynamics simulations (2.0 ns for each simulation with a 1.0-fs time step). This theoretical model was released at the Protein Data Bank (PDB; code: 1P76) before the release of the first X-ray structure of CCP (PDB code: 1Q2W). In contrast to the catalytic dyad observed in X-ray structures of CCP and other coronavirus cysteine proteinases, a catalytic triad comprising Asp187, His41, and Cys145 is found in the theoretical model of the substrate-bound CCP. The simulations of the CCP complex suggest that substrate binding leads to the displacement of a water molecule entrapped by Asp187 and His41, thus converting the dyad to a more efficient catalytic triad. The CCP complex structure has an expanded active-site pocket that is useful for anti-SARS drug design. In addition, this work demonstrates that multiple molecular dynamics simulations are effective in correcting errors that result from low-sequence-identity homology modeling.     

Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum

The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen.     

Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z-Phe-Arg-Amc (Z, benzyloxycarbonyl; Amc, 7-amidomethylcoumarin) as substrate, was fractionated by gradient ion-exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury-agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possessed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase; it was activated by dithiothreitol and EDTA and inhibited by the mechanism-based inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val-Arg-Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.     

Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase

The alpha kinases are a widespread family of atypical protein kinases characterized by a novel type of catalytic domain. In this paper the peptide substrate recognition motifs for three alpha kinases, Dictyostelium discoideum myosin heavy chain kinase (MHCK) A and MHCK B and mammalian eukaryotic elongation factor-2 kinase (eEF-2K), were characterized by incorporating amino acid substitutions into a previously identified MHCK A peptide substrate (YAYDTRYRR) (Luo X. et al. (2001) J. Biol. Chem. 276, 17836-43). A lysine or arginine in the P+1 position on the C-terminal side of the phosphoacceptor threonine (P site) was found to be critical for peptide substrate recognition by MHCK A, MHCK B and eEF-2K. Phosphorylation by MHCK B was further enhanced 8-fold by a basic residue in the P+2 position whereas phosphorylation by MHCK A was enhanced 2- to 4-fold by basic residues in the P+2, P+3 and P+4 positions. eEF-2K required basic residues in both the P+1 and P+3 positions to recognize peptide substrates. eEF-2K, like MHCK A and MHCK B, exhibited a strong preference for threonine as the phosphoacceptor amino acid. In contrast, the Dictyostelium VwkA and mammalian TRPM7 alpha kinases phosphorylated both threonine and serine residues. The results, together with a phylogenetic analysis of the alpha kinase catalytic domain, support the view that the metazoan eEF-2Ks and the Dictyostelium MHCKs form a distinct subgroup of alpha kinases with conserved properties.     

Amino acid sequence of rat mast cell protease I (chymase)

The amino acid sequence has been determined for rat mast cell protease I (RMCP I), a product of peritoneal mast cells. The active enzyme contains 227 residues, including three corresponding to the catalytic triad characteristic of serine protease (His-57, Asp-102, and Ser-195 in chymotrypsin). A computer search for homology indicates 73% and 33% sequence identity of RMCP I with rat mast cell protease II from mucosal mast cells and bovine chymotrypsin A, respectively. When the structure of RMCP I is compared to those of cathepsin G from human neutrophils and two proteases expressed in activated lymphocytes, 48-49% of the sequences are identical in each case. RMCP I has six half-cystine residues at the same positions as in RMCP II, cathepsin G, and the two lymphocyte proteases, suggesting disulfide pairs identical with those reported for RMCP II. A disulfide bond near the active site seryl residue and substrate binding site, present in pancreatic and plasma serine proteases, is not found in RMCP I or in the other cellular proteases. These results indicate that RMCP I and other chymotrypsin-like proteases of granulocyte and lymphocyte origin are more closely related to each other than to the pancreatic or plasma serine proteases.     

Definition of the extended substrate specificity determinants for beta-tryptases I and II.

Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here.     

Engineering ascorbate peroxidase activity into cytochrome c peroxidase

Cytochrome c peroxidase (CCP) and ascorbate peroxidase (APX) have very similar structures, and yet neither CCP nor APX exhibits each other's activities with respect to reducing substrates. APX has a unique substrate binding site near the heme propionates where ascorbate H-bonds with a surface Arg and one heme propionate (Sharp et al. (2003) Nat. Struct. Biol. 10, 303-307). The corresponding region in CCP has a much longer surface loop, and the critical Arg residue that is required for ascorbate binding in APX is Asn in CCP. In order to convert CCP into an APX, the ascorbate-binding loop and critical arginine were engineered into CCP to give the CCP2APX mutant. The mutant crystal structure shows that the engineered site is nearly identical to that found in APX. While wild-type CCP shows no APX activity, CCP2APX catalyzes the peroxidation of ascorbate at a rate of approximately 12 min (-1), indicating that the engineered ascorbate-binding loop can bind ascorbate.